RNA-binding protein RBM3 intrinsically suppresses lung innate lymphoid cell activation and inflammation partially through CysLT1R

Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3−/− and Rbm3−/−Rag2−/− mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3−/− ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3−/− lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3−/−Cyslt1r−/− mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R.

Lung innate lymphoid cells (ILC) are critical players in inflammatory diseases including helminth infections, asthma, and pulmonary fibrosis [1][2][3][4] . Once activated by epithelial cytokines, such as TSLP, IL-33, IL-25, and lipid mediators including leukotrienes, group 2 innate lymphoid cells (ILC2) produce type 2 cytokines IL-4, IL-5, IL-9, and IL-13 5,6 . These cytokines are major contributors to the characteristics of type 2 asthma such as airway inflammation, hyperresponsiveness, and remodeling. IL-13 promotes mucus production and airway hyperresponsiveness, IL-4 regulates IgE synthesis and Th2 cell differentiation, and IL-5 controls the survival and activation of eosinophils 7,8 . In addition to the contribution of ILC2s in asthma, there is also evidence that IL-17A production by "inflammatory" iILC2s (or ILC2-17s) as well as ILC3s promote lung inflammatory responses in asthma models 2,9,10 . For example, cysteinyl leukotrienes induce IL-17A from ST2 + lung ILC2s and transferred IL-17 −/− ILC2s are less pathogenic during type 2 lung inflammation 10 . Since the discovery of ILCs, most studies have focused on mechanisms of ILC activation, and there are fewer reports that provide insights into suppression of ILC responses. Modulation of ILC2s may occur through individual cytokines 11,12 and lipid mediators, and cell-cell contact pathways 13,14 . Interestingly, despite the known regulatory activity of microRNAs (miRNAs) in post-transcriptional repression, studies have demonstrated that miR-19 and miR-155 promote ILC2 activation and survival 15,16 . Overall, our understanding of mechanisms that broadly suppress both type 2 and 17 cytokine production in lung ILCs is limited.
Regulation of immune cell responses occurs through gene expression as well as post-transcriptional and post-translational pathways. RNA-binding proteins (RBPs) regulate cellular responses via stabilization or degradation of mRNA transcripts as well as effects on miRNA processing 17 . The RBP HuR stabilizes target mRNAs including gata3 transcripts in CD4 + Th2 cells through biding to AU-rich element (ARE) sequences in the 3'UTR 18 . AREs are also present in Th2-cytokine transcripts including Il4, Il5, Il13, and therefore are potential posttranscriptional regulatory targets for RBPs 19 . Very recently, naïve ILC2s were shown to express the RBP tristetraprolin (TTP) which inhibits Th2 cytokine production and is downregulated after IL-33 stimulation 20 . Aside from these studies, how RBPs control ILC responses is not well explored and likely represents an important level of ILC regulation in inflammatory diseases.
In this work, we perform RNA sequencing of purified lung ILC subsets from fungal allergen-challenged mice based on CD127 and ST2 21 and assess levels of RBP transcripts in ILC subsets. The Rbm3 transcript which encodes RNA-binding motif 3 (RBM3) is one of the most highly expressed RBPs in ILCs, second to Zfp36 (encodes TTP) 20 , and with a higher expression level than Elavl1, which encodes for HuR. RBM3 is a cold shock protein that has been demonstrated to enhance the stability and translation of mRNAs for COX-2, IL-8, and VEGF 22 . Further, RBM3 interacts with microRNAs miR-142-5p and miR-143, temperature-sensitive microRNAs implicated in the fever response 23 , as well as with the RBP HuR 22 . Using fungal allergen-driven and IL-33driven lung inflammation models with Rbm3 −/− , Rbm3 −/− Rag2 −/− , and Rbm3 −/− Cyslt1r −/− mice, as well as in vitro studies, mixed bone marrow chimera studies, and transcriptomic analysis, we show that RBM3 negatively regulates lung ILC type 2 and 17 cytokine responses that are partially dependent on CysLT1R signaling. Contrary to previous reports showing stabilization of cytokine transcripts by RBM3, the data presented herein demonstrates that RBM3 suppresses ILC2 activation in the lung.

Results
RBM3 is highly expressed among RNA-binding protein mRNAs in activated lung ILCs ILC subtypes can be defined by expression of CD127 (IL-7R) and ST2 (IL-33R) and we recently showed that single negative or double negative "unconventional" subpopulations for these markers include heterogenous ILC populations that express GATA-3 and produce type 2 cytokines 21 . Upon airway challenge with the asthma-associated fungal allergen Alternaria alternata, all four Thy1.2 + subpopulations (CD127 + ST2 + , CD127 + ST2 − , CD127 − ST2 + , and CD127 − ST2 − ) are activated. Lin − Thy1.2 + lymphocytes from WT mice challenged over 10 days with 50 µg Alternaria were FACS purified based on expression of CD127 and ST2 ( Supplementary Fig. 1a). Next, we performed RNA-Seq analysis and detected 207 RNA-binding protein (RBPs) transcripts expressed by the ILC subpopulations ( Supplementary Fig. 1b). Of the top 25 differentially expressed RBPs (Fig. 1a), Zfp36, which encodes for tristetraprolin (TTP), was the most highly expressed RBP. Zfp36-/-mice develop spontaneous prominent inflammation and severe autoimmune disease 24 , and a very recent report demonstrates that TTP regulates ILC2 homeostasis 20 . After Zfp36, Rbm3 was the next highest RBP with transcript levels increased over Elavl1, which encodes for HuR (Fig. 1b) which has been shown to promote GATA-3 expression and type 2 cytokines in T cells 18,25 . Since we were interested in RBP function using in vivo asthma models, we focused on regulation by RBM3 given that Rbm3 −/− mice are viable and not known to spontaneously have immune disease 25,26 . Sorted Thy1.2 + lung ILCs from Alternaria-challenged mice expressed Rbm3 mRNA at levels on par with Il5 mRNA though less than Gata3, Rora, and Il13 (Fig. 1c). qPCR analysis of the four ILC populations based on ST2 and CD127 showed that Rbm3 expression was highest in ST2 + ILCs that also more highly expressed Cysltr1, Il5, Il13, and Il17 ( Supplementary Fig. 2). Thus, of 207 RBP transcripts from in vivo activated lung ILCs, Rbm3 was expressed at levels comparable to molecules involved in ILC function and thus is a candidate protein that might regulate ILC function and lung inflammation.

Alternaria, IL-33, and TSLP increase lung and ILC RBM3 expression
To ascertain whether RBM3 is induced during type 2 inflammation, Alternaria-challenged mice were assessed for RBM3 levels by immunostaining. Increased expression of RBM3 was detected by immunofluorescence in Alternaria-challenged lungs compared to naïve control lungs ( Supplementary Fig. 3a). RBM3 expression was visualized in epithelial as well as subepithelial cells which was further induced by challenge with Alternaria. As ILCs regulate innate lung responses to Alternaria 26 , we investigated changes in RBM3 levels in lung ILCs. Lin − Thy1.2 + ILCs from the lungs of challenged mice demonstrated increased RBM3 expression by intracellular flow cytometry when compared to naïve and PBS-challenged controls (Fig. 1d). Interestingly, eosinophils and macrophages analyzed from challenged mice did not show increases in RBM3 expression ( Supplementary Fig. 3b).
As IL-33 and TSLP are critical epithelial cytokines that regulate ILC2 responses, we assessed levels of RBM3 in Tslpr −/− mice and WT mice treated with IL-33 blocking antibody. RBM3 expression in Tslpr −/− mice and WT mice receiving anti-IL-33R was reduced compared with controls ( Fig. 1e). We next assessed whether human ILC2s expressed RBM3 and if TSLP and IL-33 regulated RBM3 expression as they do in mouse ILCs. FACS purified human ILC2s stimulated with a combination of TSLP and IL-33 demonstrated significantly increased RBM3 immunostaining after 24 h and 72 h compared with control staining (Supplementary Fig. 4). Thus, activation of ILCs by epithelial cytokines IL-33 and TSLP led to increased RBM3 expression in both mice and humans, suggesting studies of RBM3 in mouse ILCs might be relevant to humans.

RBM3 suppresses Alternaria-induced type 2 lung inflammation
We next performed in vivo asthma model experiments with Rbm3 −/− mice to assess lung inflammatory and ILC responses. We analyzed lineage-negative Thy1.2 + lung ILCs from naïve Rbm3 −/− mice and found no significant changes in the absolute or relative number of ILCs between naïve WT and Rbm3 −/− mice ( Supplementary Fig. 5a, b). Naive Rbm3 −/− ILCs had similar surface marker expression of common ILC2 surface markers when compared to WT ILC2s ( Supplementary  Fig. 5c), and naïve Rbm3 −/− mice also showed similar levels of BAL and lung eosinophils as WT controls ( Supplementary Fig. 5d, e). Thus, Rbm3 −/− mice are phenotypically similar to WT mice in terms of baseline lung eosinophil and ILC levels.
The effects of global RBM3 deficiency were not limited to ILCs. After Alternaria-challenges, the total number of ST2 + CD4 + T cells in BAL and lung were greater in Rbm3 −/− mice compared to WT control ( Supplementary Fig. 6a). Ki-67 expression within ST2 + T cells was also significantly increased in Rbm3 −/− mice. ST2 expression within T cells also trended higher in Rbm3 −/− mice lung and BAL ( Supplementary  Fig. 6b, c). Thus, it is plausible that RBM3 also directly or indirectly suppresses CD4 + Th2 cell responses.
( Fig. 4a-c). BAL and lung eosinophils were also significantly increased in the double knock-out mice compared to controls (Fig. 4d). Overall, these results show a suppressive effect of RBM3 in type 2 lung inflammation and ILC activation, even in the absence of adaptive immunity.

RBM3 directly suppresses IL-33-induced ILC responses in vitro and in vivo
To establish whether RBM3 directly regulated ILC responses, we performed studies with IL-33-stimulated ILCs in vitro and in vivo. challenged mice were rested for 48 h in media alone prior to stimulation. Immediately prior to stimulation, Rbm3 −/− ILC2s produced significantly more IL-5 by ELISA than wild-type ILC2s (Fig. 5a). In contrast, there were no differences in the production of IL-13 prior to stimulation. IL-33 stimulation (15 ng/ml) of Rbm3 −/− ILCs resulted in increased IL-5 and IL-13 compared with wild-type ILCs (Fig. 5b). ILCs lacking RBM3 produced significantly more IL-5 after stimulation with IL-33 (both 15 and 30 ng/ml IL-33). Consistent with data in Fig. 3d, ILC IL-17A production was increased in Rbm3 −/− ILCs after 48 h of rest (Fig. 5c). Unlike the Type 2 cytokines, IL-17A was not further increased above pre-stimulation conditions with IL-33 stimulation (Fig. 5c). These results demonstrate a direct inhibitory regulatory function of RBM3 in ILC type 2 cytokine production in response to IL-33, as well as a suppressive effect of RBM3 in IL-17A production measured ex vivo.

Discussion
Innate lymphoid cells including ILC2s have recently emerged as critical contributors to immune-mediated diseases including asthma 40,41 . The majority of the ILC literature thus far has reported soluble factors that activate or inhibit ILC2 function including cytokines and lipid mediators 42 . However, an understanding of intracellular mechanisms that more broadly control ILC function might provide important insights into ILC-driven immune diseases. Studies thus far have demonstrated that specific miRNAs activate ILC2s through multiple mechanisms 15,16 . Here, we found that RBM3 is a highly expressed RNA-binding protein (RBP) in lung ILC subsets which is induced by epithelial cytokines IL-33 and TSLP, and negatively regulates ILC type 2 and 17 cytokine production. Mixed bone marrow congenic chimera mice displayed increased type 2 cytokine production in ILCs from Rbm3 −/− mice, highlighting an ILC-intrinsic activity of RBM3. Further, transcriptomic analysis demonstrated broad differences in Rbm3 −/− ILCs including expression of CysLT1R, which we found to regulate ILC2 IL-17 + cell and eosinophil accumulation in Rbm3 −/− mice. RBM3 is a 17KD RNA-binding protein that promotes mRNA stability and translation efficiency by binding to ARE binding regions. Very little is understood about RBM3 in inflammation and immunity. One report showed that RBM3 is downregulated in febrile illness and knockdown of RBM3 led to increases in miRNAs that suppress PGE2, IL6, and IFNA1 23 . However, earlier studies showed that RBM3 deficient mice had normal numbers of NK, T, and B cells and had no differences in innate cytokine responses to the TLR9 ligand CpG 43 . Stressors such as tissue hypoxia and low temperatures can induce RBM3 expression 44 . Our work demonstrates that RBM3 is induced by IL-33 and/or TSLP in mouse and human ILC2s. Of note, IL-33 has been reported to promote a tumor hypoxic microenvironment, with generation of reactive oxygen species, that could lend toward an indirect induction of RBM3 through local hypoxia 45,46 . Further, in addition to hypoxia and hypothermia, activation of NF-κB promotes RBM3 expression and extracellular IL-33 activates NF-κB 47,48 . Thus, there may be multiple mechanisms by which IL-33 and/or TSLP may induce RBM3 expression under inflammatory stress conditions. We found that RBM3 suppresses the classic ILC2 cytokines (IL-5 and IL-13) as well as IL-17A and ILC proliferation. Furthermore, exacerbation of type 2 responses in Rbm3 −/− mice was independent of changes in total ILC GATA3 levels and lacked correlation with number of ILC AU-rich element (ARE) transcripts. RBM3 has a multitude of complex potential mechanisms that regulate cellular changes during stress including controlling translation efficiency through ARE binding, protein-protein interactions, and effects on miRNAs directly or through dicer processing 17,23,44 . Interestingly, RBM3 has also been reported to inhibit the p38 MAP kinase pathway which promotes cytokine production by ILC2s in response to IL-33 [49][50][51] . Thus, removal of RBM3's inhibition of the p38 pathway in ILCs may result in increased cytokine production in Rbm3 −/− ILCs. Our studies also demonstrate that RBM3 has no effect on ILC2 numbers and lung eosinophilia under homeostatic naïve conditions but has a clear suppressive function during type 2 inflammatory insult. This is consistent with RBM3 being previously identified as a "stress-response" protein that is largely protective in neural survival in conditions of stress 44 . Our data suggest that RBM3 limits hyperactive ILC responses which could be an important protective mechanism during lung inflammation.
iILC2s (or ILC2-17s) induced by IL-25, cysteinyl leukotrienes, and Notch signaling 9,10,53 . Initial reports showed that ST2-negative ILC2s were "inflammatory" ILCs that produce IL-17A in response to IL-25 9 . However, Cai et al. subsequently reported that ST2 + ILC2s are also key IL-17A producers (known as ILC2-17s) which can also be induced by CysLTs 10 . Similarly, dual cytokine staining in our studies showed that ILC2s were the dominant ILC source of IL-17 in our models. Despite these results, we cannot exclude the presence of independent populations of ILC3s or other IL-17A-producing ILCs that are regulated by Rbm3 −/− mice in different contexts.
RBM3 is expressed in multiple immune cells, including eosinophils, macrophages, and T cells. However, we investigated the direct Article https://doi.org/10.1038/s41467-022-32176-5 Fig. 6 | Mixed bone marrow chimeras show cell-intrinsic exaggeration of a type 2 response in ILCs from Rbm3 −/− mice. a Schematic of bone marrow reconstitution. Mixed bone marrow chimeras were generated using bone marrow from CD45.1 Rbm3 +/+ (WT) mice and CD45.2 Rbm3 −/− mice and reconstituted for 10-12 weeks, images created with BioRender.com. b Mixed bone marrow chimera mice were challenged 3 times over 7 days with 20 μg Alternaria. FACS plots are representative of 2 independent experiments (5 mice each, n = 10) with biological controls. ILCs are gated as Lineage − Thy1 . 2 + cells. c Percent (p = 0.0178) and total (p = 0.0003) IL5 expressing ILCs from WT vs. Rbm3 −/− mice. FACS plots of IL5 percentages. d Percent (p = 0.0012) and total IL13 (p = 0.0029) expressing ILCs from WT vs. Rbm3 −/− mice. Representative FACS plots of IL13 percentages. e Percent (p = 0.7764) and total Ki67 (p = 0.0001) expressing ILCs from WT vs Rbm3 −/− mice. Representative FACS plots of Ki67 percentages. Paired t-Test, two-tailed. *p < 0.05, **p < 0.005, ***p < 0.0005. function of RBM3 in lung ILCs given the emerging functions of ILCs during lung inflammation. In the mixed bone marrow chimera studies, we show that Lin − Thy1.2 + Rbm3 −/− ILCs were more activated compared with congenic WT ILCs. In contrast, Lin + Thy1.2 + populations, which largely include T cells, did not demonstrate a significant change in proliferation of type 2 cytokine production between WT and Rbm3 −/− cells. Thus, it appears that there may be a more selective intrinsic contribution of RBM3 in ILCs during type 2 inflammation compared with T cells. However, Th2 cells were increased in the complete Rbm3-/mice after Alternaria challenges and this may be due to early ILC2 contributions to adaptive Th2 responses as previously reported 54,55 . To explore potential mechanisms by which RBM3 regulates ILC biology, we performed transcriptomic studies which demonstrated global changes in activation of ILCs by RBM3 including differential expression of known ILC cytokines, receptors as well as transcription factor, and survival transcripts. Importantly, cytokine transcript data from Rbm3 −/− ILCs supported our in vivo findings that RBM3 suppressed type 2 and IL-17A cytokine production from ILCs. Transcripts for multiple receptors were increased in Rbm3 −/− ILCs, including Il1rl1 (encodes ST2), Il7r, Il2rg, Cysltr1, and Cd44. Though Il1rl1 was increased at a transcript level, we did not detect increased ST2 at a protein level by flow cytometry. Notably, several ILC transcription factors were increased in Rbm3 −/− ILCs including Tox, Ets1, Rora, Irf4, and Id2 1,29-33 . RORα and ETS1 also promote ILC2 cytokine production suggesting ILC2 cytokine production could be regulated by RBM3 through control of these transcription factors 30,56 . Despite differences in levels of developmental transcription factors in Rbm3 −/− ILCs, we did not detect differences in lung ILCs in naive Rbm3 −/− mice. This may be explained by induction of RBM3 in inflammatory settings which exerts effects on mature ILCs while being dispensable for ILC development.
Increased expression of the Cysltr1 transcript in Rbm3 −/− ILCs was a particularly interesting finding because it was accompanied by an increase in expression of Nfat2c (encodes NFAT1). Previous reports have shown that CysLT1R signaling in ILCs is regulated by NFAT1 to promote increased Th2 and Th17 cytokine production 10,38,57,58 . Therefore, we generated Rbm3 −/− Cyslt1r −/− double knockout mice, which showed that CysLT1R regulates some of the hyperinflammatory phenotype observed in Rbm3 −/− mice after Alternaria challenges. Interestingly, ST2 + IL-17 + ILCs were also controlled by CysLT1R in Rbm3 −/− mice which is in line with previous data that CysLTs induce IL-17 from ST2 + ILCs 10 . As ILC2 responses through CysLT1R are regulated by NFAT1, RBM3 suppression of the CysLTR1/NFAT1 axis could potentially limit ILC-driven lung inflammation though it is likely that other RBM3dependent mechanisms contribute as well.
In summary, this work identifies an important function of RBM3, a highly expressed RNA-binding protein, in activated lung ILCs that is induced by IL-33 and TSLP. RBM3 dampens both type 2 cytokine and IL-17A cytokine production by lung ILCs and decreases downstream granulocyte infiltration in the setting of fungal allergen and IL-33 exposure. Mixed bone marrow chimera studies highlighted the cellintrinsic effect of RBM3 on ILC function and transcriptomic analysis demonstrated RBM3 regulation of multiple cytokines, transcription factors, survival genes, and receptors critical to ILC function including CysLT1R. Studies of allergen-challenged Rbm3 −/− Cyslt1r −/− double knockout mice demonstrated that CysLTR1 contributes to RBM3mediated ILC responses. The work presented herein further substantiates RNA-binding proteins as critical post-transcriptional regulatory mediators of ILC activation during type 2 inflammation.

Methods
The research complies with all relevant ethical regulations and was approved by the UC San Diego IACUC animal care committee. Reagents for flow cytometry, qPCR, ELISA, allergen, and cytokines detailed in the Supplementary Data file.
Mice 6-12-week-old female and male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). Wild-type mice were age and gender matched to Rbm3 −/− mice acquired from Peter Vanderklish at TSRI and originally from Tadatsugu Taniguchi 43 at University of Tokyo and bred in house. Tslpr −/− mice were acquired from Dr. Michael Croft at the La Jolla Institute for Immunology and originally from Dr. Steven Ziegler 59 . The Rbm3 −/− Rag2 −/− mice were created through multiple crosses of Rbm3 −/− and Rag2 −/− mice and bred in house. The Rbm3 −/− Cyslt1r −/− mice were created through seven crosses of Rbm3 −/− and Cyslt1r −/− mice (Jax stock number #030814) and bred in house. Mixed bone marrow chimera studies utilized CD45.1 + PEP boy homozygotes and CD45.1 + CD45.2 + PEP boy heterozygotes (Jax stock number: #002014). WT and Rag2 −/− (Jax stock number #008449) mice originated from Jackson labs (Bar Harbor, ME). All mice were on a C57BL/6 background and controls were age and gender matched. All studies were approved by the University of California, San Diego Institutional Animal Care and Use Committee.
previously reported 21 . Hematoxylin and Eosin (H&E) and Periodic acid-Schiff (PAS) staining were performed at the Histology Core in UCSD's Moore's Cancer Center and imaged with microscopy as previously reported 21 . H&E and PAS stained slide images were captured at 20X magnification for levels of peribronchial inflammation and PAS stained slides, respectively.

ELISA
Samples stored at −20C were analyzed using IL-5 and IL-13 ELISA kits (R&D Systems, Minneapolis, MN) per the company's instructions. Plates were read using a microplate reader model 680 (Bio-Rad Laboratories, Hercules, CA). ELISA data was analyzed using Excel and Graphpad Prism (San Diego, CA).

ILC purification and RNA Sequencing
WT and Rbm3 −/− ILCs or WT ILCs based on ST2 and CD127 expression were sorted with the BD FACSAria II or the BD FACSAria Fusion at the UCSD Human Embryonic Stem Cell Core Facility. Lin − Thy1.2 + ILCs were sorted directly into TrizolLS. RNA-sequencing was performed at the La Jolla Institute. RNA-sequencing data of Rbm3 −/− and WT ILCs was newly generated for this study and deposited in GEO database (GSE155330). RNA-sequencing of ILCs based ST2 and CD127 were previously reported 21 and is deposited in GEO database (GSE136156).
Briefly, purified total RNA (≈5 ng) was amplified following the Smart-seq2 protocol 60,61 . mRNA was captured using poly-dT oligos and directly reverse-transcribed into full-length cDNA using the described template-switching oligo 60,61 . cDNA was amplified by PCR for 15 cycles and purified using AMPure XP magnetic bead (0.9:1 (vol:vol) ratio, Beckman Coulter). From this step, for each sample, 1 ng of cDNA was used to prepare a standard NextEra XT sequencing library (NextEra XT DNA library prep kit and index kits; Illumina). Barcoded Illumina sequencing libraries (NextEra; Illumina) were generated utilizing an automated platform (Biomek FXP, Beckman Coulter). Both wholetranscriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality control steps were included to determine total RNA quality and quantity, the optimal number of PCR preamplification cycles, and fragment library size. The reference genome was mm10 (mouse genome). None of the samples failed quality controls. All of the samples were pooled at equimolar concentration, loaded, and sequenced on the Illumina Sequencing platform, HiSeq2500 (Illumina). Libraries were sequenced to obtain more than 10 million 50-bp single-end reads (HiSeq Rapid Run Cluster and SBS Kit V2; Illumina) mapping uniquely to mRNA reference.
For cytokine intracellular staining of lung cells in the 10-day challenge model, cells were cultured overnight with Golgi Plug (Fisher Scientific, Hampton, NH) at 500,000 cells per well. After surface staining for ILCs, cells were fixed and permeabilized using the BD fixation/permeabilization kit (BD Biosciences, La Jolla, CA) and stained for IL-5 (PE) or IL-13 (PE). For cytokine intracellular staining following the 7-day IL-33 and Alternaria challenge model, lung cells were cultured for 3 h with cell stimulation cocktail (ThermoFisher, Waltham, MA) at 1 × 10 6 cells per well. After surface staining for ILCs, cells were fixed and permeabilized using the BD kit and stained for IL-5 (PE), IL-13 (PE), or IL17A (eFlour506). Lung cells stained for Bcl-2 expression were surfaced stained for ILCs, fixed and permeabilized with the BD kit, and stained with anti-Bcl-2 (PE-Cy7).
For human PBMC staining, ILC2s were sorted as Lineage − CRTH2 + lymphocytes. The lineage cocktail (FITC) consisted of antibodies for CD3, CD14, CD16, CD19, CD20, CD56, TCRγδ, CD4, CD11b, CD235a, and FcεRI. The polyclonal RBM3 antibody used in this study was raised in rabbits to the 14 c-terminal amino acids of RBM3, and affinity purified to the immunizing peptide. As described in prior work 62,63 , the affinitypurified anti-RBM3 antibody recognizes an~17 kDa band corresponding to RBM3 on Western blots and selectively labels RBM3 in situ under a variety of fixation conditions. Flow Cytometry was performed using the BD Accuri for Fig. 1 and Supplemenatary Fig. 3b, otherwise, Acea Novocyte was used. Data was analyzed using FlowJo software (Tree Star, Ashland, OR). All antibodies were from Biolegend, ThermoFisher, or BD Biosciences.

Immunofluorescence
For mouse airways, immunofluorescence for RBM3 was performed on naïve and Alternaria challenged airways as previously reported 64,65 . Briefly, lung samples were de-paraffinized by sequential placement in xylene and ethanol. Staining for RBM3 was performed with rabbit polyclonal antibody (PeproTech) at 1:1000 concentration. Tyramide Signal Amplification Kit #41 (Invitrogen) was used for fluorescent signal amplification with subsequent DAPI staining (Vector Laboratories). Lung airways were visualized with a DM2500 microscope (Leica Microsystems). Cell nuclei were stained with DAPI. Images were taken from at least 5 airways of at least 3 mice per group.

ILC and PBMC cultures
WT and Rbm3 −/− Lin − Thy1.2 + ILCs were sorted using the BD FACSAria Fusion and BD FACSAria II sorters from UCSD's Human Embryonic Stem Cell Core Facility. Collected ILCs were rested in 10 ng/mL IL-2 and IL-7 (R&D Systems, Minneapolis, MN) in T cell media (RPMI + 10% FBS, 1% glutamine, 0.1% BME, 1% pen/strep) for 48 h. ILCs were cultured in a 96-well plate at 40,000 cells per well. Pre-stimulation media was collected and stored in −80C for ELISA. ILCs were stimulated for 24 h with 15 ng/mL or 30 ng/mL IL-33 (R&D Systems, Minneapolis, MN) in T cell media. Post-stimulation supernatant was collected and stored at −80 C for ELISA analysis.
Human PBMCs were isolated from commercially purchased leukopacks (Allcells, Alameda, CA, USA). Human peripheral blood ILC2s were sorted as CD45 + lin − CRTH2 + lymphocytes and cultured and treated with TSLP and IL-33 before being fixed with 4% PFA and processed for immunocytochemistry using a RBM3 antibody (1:2000) and a Cy3 secondary. Cells were also stained for DAPI. Images were taken at 20X. Immuno-positive elements were captured and analyzed by thresholding the intensity histogram in the Cy3 channel at 100. Data for objects of 50-175 pixels were included.

Statistical analysis
Statistical analysis was performed with GraphPad Prism software (GraphPad Software, La Jolla, CA). P-values were obtained using the Mann-Whitney test, unpaired t-test, or one-way ANOVA and a P value of less than 0.05 was considered statistically significant such that *p < 0.05, **p < 0.01, ***p < 0.001.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Reagents for flow cytometry, qPCR, ELISA, allergen, and cytokines are detailed in the Supplementary Data file. RNA-sequencing data of Rbm3 −/− and WT ILCs was newly generated and deposited in GEO database (GSE155330). RNA-sequencing of ILCs based ST2 and CD127 was previously reported 21 and is deposited in GEO database (GSE136156). There are no restrictions with obtaining unique biological materials which are available from the corresponding author upon reasonable request. Source data are provided with this paper.